The long-term objective of this project is to develop a comprehensive molecular diagnostic assay for the detection of clinically relevant genetic abnormalities in patients with acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and myeloproliferative disorders (MPDs). An estimated 44,000 new leukemia cases were predicted for 2008, with more than 21,000 deaths. Leukemia and MPDs are a group of similar diseases of the immune system that have different symptoms, treatment targets, and prognoses. Risk stratification for these diseases has shifted from morphology and cytogenetic analysis to molecular markers. The variation in clinical outcome based on disease subtype underscores the very real need to ascertain for each individual patient the combined cytogenetic and mutational events that underlie the disease;thus, these markers are used as an aid in diagnosis. Furthermore, the products of these cytogenetic and mutational alterations are being exploited as molecular targets for novel therapies. Thus, these treatments will require companion diagnostics for comprehensive detection of molecular signatures that are predictive of treatment outcome. During Phase I we established the feasibility of detecting a small set of mutations with clinical relevance to leukemias and MPDs by demonstrating specific detection of a small set of mutations using a multiplex bead array system. Furthermore, we demonstrated simultaneous detection of mutations and translocations in a single-tube assay format. This resulted in the launch of an RUO diagnostic kit for the detection of NPM1 mutations (Signature(R) NPM1 Mutations). The primary goal of this Phase II grant is to expand the assay into a comprehensive system for simultaneous detection of a suite of sequence-level mutations and cytogenetic abnormalities. In addition to expanding the availability of molecular assays for leukemia/MPD testing, our single-tube, bead array format will simplify adoption into diagnostic laboratories in a manner that reduces overall assay time, operator hands-on time, and training time and provides a software interface that simplifies reporting and facilitates reimbursement. We will develop a set of synthetic controls as well as a software interface for test selection and reporting and perform stability testing and other assay performance experiments. We will also work with clinical collaborators to evaluate the test in a diagnostic laboratory setting on clinical patient samples. PUBLIC HEALTH RELEVANCE: The goal of this Phase II SBIR project is to develop a reliable and cost-effective diagnostic test kit for mutations found in leukemia patients. Detection of mutations using our test will allow physicians and patients to choose the most effective treatment for each individual patient while improving efficiency and driving down costs in the molecular diagnostic laboratory.